Platelet glycoprotein VI (GPVI), a 62kD membrane protein, has been identified as one of the platelet receptors for collagen, since GPVI-deficient platelets exhibit abnormal responses to collagen and an abnormal bleeding tendency. We report a female patient with a mild bleeding history whose platelets expressed 10% GPVI of normal platelets. Shape change, aggregation and ATP release of the patient's platelets were completely absent in response to 1-5 micrograms/ml collagen but present normally in response to ADP and Ca2+ ionophore A23187. Adhesion of the patient's platelets to coated collagen was mildly affected (40-60% of normal platelets) in spite of only 10% expression of GPVI. Flow cytometrical studies revealed that the patient's platelets expressed normal amounts of the GPIa/IIa complex. These results suggest that platelet GPVI is less involved in adhesion to collagen than shape change and aggregation induced by collagen.

The accumulation of monocyte/macrophages and T lymphocytes in arterial intima is a hallmark of early atherogenesis. To investigate the temporal relationships between endothelial expression of adhesion molecules (eg, P-selectin and vascular cell adhesion molecule-1 [VCAM-1]) and intimal accumulation of macrophages and T lymphocytes, immunostaining was performed by using serial frozen sections from intercostal branch points of thoracic aortas of New Zealand White rabbits that had been fed a 0.3% cholesterol diet. After 1 week of cholesterol feeding, neither macrophages nor T lymphocytes were detected, although endothelial expression of P-selectin and VCAM-1 was observed. After 3 weeks, macrophages were detectable in 75% and T lymphocytes were present in 25% of the rabbits. Expression of P-selectin and VCAM-1 was sustained until 10 weeks. Infiltration of T lymphocytes was restricted in areas in which macrophages were accumulated and did not appear to precede macrophage infiltration. E-selectin expression was not detectable before accumulation of mononuclear leukocytes; however, very few endothelial cells covering foam cell lesions expressed E-selectin after 6 weeks. Similar results were obtained in Watanabe heritable hyperlipidemic rabbits aged 1, 2, and 3 months. Taken together, localized expression of P-selectin and VCAM-1 may play a key role in the initial recruitment of macrophages and T lymphocytes in early atherogenesis.

The individual contributions of glycoprotein Ib (GPIb) and the seven transmembrane domain receptor (STDR) to increases in platelet [Ca2+]i induced by alpha-thrombin or the tethered ligand peptide (TLP; SFLLRNPNDKYEPF) have been determined in control platelets, in platelets where the thrombin binding site on GPIb was blocked with the monoclonal antibodies TM60 and LJ-Ib10, in platelets where access of thrombin to the STDR was blocked by polyclonal antipeptide antibodies, and in Bernard-Soulier platelets which constitutively lack GPIb. Curve-fitting analyses (LIGAND) showed that binding of PPACK-thrombin and alpha-thrombin to the moderate-affinity site was not detected in the best-fit model in the presence of anti-STDR antibodies although with alpha-thrombin there was also decreased binding at the high-affinity site. Conversely, TM60 blocked binding of alpha-thrombin to the high-affinity site but also decreased binding at the moderate affinity site. Separately, either TM60 or anti-TNA (150 micrograms/mL) reduced thrombin (0.5 nM)-induced elevations in [Ca2+]i to 50% of control values, but Ca2+ elevations were essentially abrogated (4.2 +/- 5%) when the two were added in combination. [Ca2+]i dose-response curves for alpha-thrombin were curvilinear and were only 50% of controls in the presence of anti-GPIb or anti-STDR antibodies at up to 10 nM alpha-thrombin, with their greatest sensitivity being below 2 nM. With Bernard-Soulier platelets, changes in [Ca2+]i were not detectable at < or = 0.5 nM alpha-thrombin but were also 50% of controls at 5-10 nM alpha-thrombin. [Ca2+]i responses to TLP (1-100 microM) of antibody-blocked platelets were identical to those of controls whereas responses were approximately 50% of controls in Bernard-Soulier platelets. The rate of increase in [Ca2+]i in controls was twice that seen in antibody-blocked platelets and about 5-fold greater than in Bernard-Soulier platelets. These results demonstrate that both GPIb and the STDR are required to ensure the optimal rate and extent of platelet activation over a range of alpha-thrombin concentrations (0.3-10 nM) and that the STDR corresponds to the previously described moderate-affinity thrombin receptor.

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